Complete Genome Sequence of Streptomyces Phage ϕRKBJ001 Isolated from Prince Edward Island, Canada

ABSTRACT We reported here the complete genome sequence of Streptomyces phage ϕRKBJ001 that was isolated from a saltwater marsh on Prince Edward Island, Canada, using the Streptomyces sp. strain RKBHB0173. Based on electron microscopy and genomic analysis, this phage belongs to the Siphoviridae family and the BN Streptomyces phage cluster.

S treptomyces spp. are a rich source of natural products (NPs), but, in the laboratory setting, the majority of biosynthetic gene clusters (BGCs) that encode these NPs remain dormant (1). As part of an effort to better understand how bacteriophages impact cryptic BGCs expression in Streptomyces bacteria, we used Streptomyces sp. strain RKBHB0173 (GenBank accession no. MW494619) to isolate phage f RKBJ001 from a sediment sample collected from a saltwater marsh (depth, 0.15 m; temperature, 26.3°C; pH, 7.2; salinity, 24.5 ppt) near Kinlock Beach on Prince Edward Island, Canada (46.199302 N, 63.0812886 W) in August of 2019.
PhiRKBJ001 was enriched using RKBHB0173 and isolated by following established protocols (2,3), with minor adjustments. Briefly, the sediment sample was centrifuged at 1000 Â g to remove excess water and a slurry of 10 g of sediment in 20 mL of phage buffer (10 mM Tris-HCl [pH 7.5], 10 mM MgSO 4 , 68 mM NaCl) was agitated at 300 RPM for 1 h to extract phage from the sediment. Samples were vortexed for 10 s and clarified by centrifugation followed by filtration (0.22 mm cellulose acetate). Extracted phage was enriched by incubating with RKBHB0173 spores at 30°C and 200 RPM for 2 days in 1:1 ratio with double strength International Streptomyces Project-2 (ISP2) growth medium (4) supplemented with 18% Instant Ocean® Sea Salt and 1 mM CaCl 2 .
Pure stocks of f RKBJ001 were obtained by 3 rounds of plaque purifications by streak plating and a high-titer stock (5 Â 10 12 PFU/mL) was generated from CsCl density-gradient ultracentrifugation. Genomic DNA was extracted with phenol-chloroform based on published protocols (3) and DNA purity and integrity were assessed spectrophotometrically and by gel electrophoresis. Library preparation and sequencing were conducted by the center d'expertise et de services at Genome Québec using the NEBNext® Ultra II DNA Library Prep kit for Illumina (NEB BioLabs) and the Illumina MiSeq platform to produce a total of 197,206 paired-end 250-bp reads. Geneious Prime software (version 2020.2.2) was used to trim the paired reads with BBDuk (version 38.84) using the following settings: kmer length = 27, minimum quality and overlap = 20, and minimum length = 50. De novo genome assembly was performed on 157,246 trimmed reads with the Geneious Assembler module using the default settings and the option to circularize contigs if ends match with 3 or more sequences selected. The 153,548 reads were assigned to one contig that was circularized into a consensus sequence with an average read coverage of 443Â. The assembled genome was annotated in Geneious Prime with the Glimmer 3 plugin (version 1.5) and with web software GeneMarkS (version 4.28) (5). The locations of start codons were adjusted based on sequence homology to related phage open reading frames (ORFs) while maintaining an overlap of less than 40 bp with the upstream ORF, if applicable. Functional ORFs were assigned based on results from the nonredundant protein sequences search database in BLASTP (6) and the default/structural domain database in HHpred (7), using default settings. Web software tRNAscan-SE (8) was used to search for the presence of tRNAs.  PhiRKBJ001 had a circularly permuted DNA genome of 69,373 bp with a GC content of 63.7%. Based on nucleotide sequence homology, f RKBJ001 clusters with the lytic Streptomyces BN cluster (Table 1) and had the closest sequence similarity to phage Wentworth. Electron micrographs confirmed that f RKBJ001 belongs to the Siphoviridae family (Fig. 1). The f RKBJ001 genome is devoid of any tRNAs, which was consistent with other phages in the BN cluster. Thirty percent of the 109 annotated ORFs were identified as functional phage proteins, while 5 of the hypothetical proteins were unique to f RKBJ001. Further studies of f RKBJ001 and its associated genes will be performed to determine their impact on the regulation of host BGCs.
Data availability. The annotated genome of f RKBJ001 was deposited in GenBank with accession no. MT936332 and the sequencing data have been deposited in the Sequence Read Archive with accession no. SRX9062343 under BioProject accession no. PRJNA660661.

ACKNOWLEDGMENTS
This work was supported by a Natural Sciences and Engineering Council of Canada (NSERC) Discovery grant and an NSERC Postdoctoral Fellowship that was awarded to BPJ.
We acknowledge the center d'expertise et de services at Genome Québec for library preparation and Illumina MiSeq sequencing services. We thank Mary Ann Trevors at the CORES EM Facility at Dalhousie University for processing and generating electron micrographs of our phage sample.
We declare no conflict of interest.